Trypanosoma cruzi (“T. cruzi”), the protozoan parasite that causes Chagas' disease, or American trypanosomiasis, is endemic in Central and South America as well as in Mexico. Most infected persons, after a mild acute phase, enter the life-long indeterminate phase that is characterized by a lack of symptoms, low parasitemias, and antibodies to a variety of T. cruzi antigens. Approximately 10-30% of persons with chronic T. cruzi infections, however, develop cardiac or gastrointestinal dysfunction as a consequence of the persistent presence of the parasite. Chemotherapy is largely ineffective, particularly for chronic infections. Roughly 25,000 of the estimated 12 million people in the endemic countries who are chronically infected with T. cruzi die of the illness each year, typically due to cardiac rhythm disturbances or congestive heart failure (See, Kirchoff, L. V., “American trypanosomiasis (Chagas' disease) in Tropical Infectious Diseases: Principles, Pathogens and Practice, R. L. Guerrant et al., editors, p. 1082-1094, Churchill Livingstone, N.Y. 2006).
In endemic areas T. cruzi is transmitted mainly by blood-sucking triatomine insects. Transmission can also occur by transfusion of blood donated by chronically infected persons, and historically this route of transmission was important in the endemic countries prior to the implementation of blood screening programs (See, Schunis, G. A., Clin. Microbiol. Rev., 18:12-29 (2000)). There is no vaccine for preventing transmission of T. cruzi. During the last few decades emigration from Chagas-endemic countries to the U.S. has increased markedly. Approximately 13 million such immigrants now live in the U.S., and an estimated 80,000-120,000 of these persons are infected with T. cruzi (See, Kirchoff, L. V., et al., Transfusion, 46:298-304 (2006)). Their presence creates a risk of transfusion-related transmission of the parasite in the U.S. Five instances of transfusion-related Chagas disease have already been reported in the U.S., and blood bank authorities agree that a much larger number of undiagnosed cases have likely occurred (See, Leiby, D. A., et al., N. Engl. J. Med., 341:1237-1239 (1999) and Young, C. T., “Transfusion acquired Trypanosoma cruzi infection,” Transfusion, In press (2007)). Currently the U.S. blood supply is not screened for T. cruzi, as no blood screening assay has been cleared by the FDA. Hence, T. cruzi infection is a threat to the U.S. blood supply. T. cruzi can also be transmitted by transplantation of organs obtained from chronically infected persons. Numerous reports of such transmission have been reported in the endemic countries, and it is unclear how many have gone undetected in the U.S. (See, Mascola, L., et al., MMWR, 55:798-780 (2006) and Zayas, C. F., et al., MMWP, 51:210-212 (2001)).
Laboratory diagnosis of chronic T. cruzi infection is complex. Demonstration of the parasite by hemoculture or xenodiagnosis is time-consuming, insensitive, and expensive. In contrast, serologic assays for antibodies to T. cruzi are well suited for rapid and inexpensive diagnosis of the infection. Conventional tests, such as indirect hemagglutination assay (“IHA”), indirect immunofluorescence assay (“IFA”), and enzyme-linked immunosorbent assay (“ELISA”), are used widely in the endemic countries. Most are based on whole or semi-purified antigenic fractions from T. cruzi epimastigotes grown in axenic culture. A persistent problem with the conventional assays has been the occurrence of inconclusive and false-positive results (Almeida, I. C., et al., Transfusion, 37:850-857 (1997), Kirchoff, L. V., et al., Transfusion, 46:298-304 (2006) and Leiby, D. A., et al., J. Clin. Microbiol., 38:639-642 (2000)). There is no consensus on which parasite antigen preparation is best for detecting antibodies to T. cruzi. The Pan American Health Organization and other expert groups have recommended that donated blood be tested by at least two different methods run in parallel (See, “Control of Chagas Disease”, World Health Organization, Geneva (2000)). This approach carries with it an enormous logistical and economic burden for blood banks.
Thus, there is a need in the art for a supplemental assay for use in clinical laboratories and blood banks. No assay has been uniformly accepted as the gold standard for the serologic diagnosis of T. cruzi infection. PCR-based assays lack the sensitivity necessary for this role (See, Gomes, M. L., Am. J. Trop. Med. Hyg., 60:205-210 (1999)). A radioimmune precipitation assay (“RIPA”), which is a highly sensitive and specific test with easily interpreted results was developed nearly two decades ago and has been suggested for use as a confirmatory test in the U.S. (See, Kirchoff, L. V., et al., J. Infect. Dis., 155:561-564 (1987)). Although the RIPA has been used as a confirmatory assay in more than 20 research projects reported to date (See, Kirchoff, L. V., et al., Transfusion, 46:298-304 (2006) and Leiby, D. A., et al., Transfusion, 42:549-555 (2002)), its sensitivity and specificity have not been systematically validated. Moreover, the complexity of the RIPA would make its widespread use outside of research settings difficult (See, Leiby, D. A., et al., J. Clin. Microbiol., 38:639-642 (2000)).
Immunoblot assays have also been studied as supplemental tests for antibodies to T. cruzi. Some years ago an immunoblot assay based on a T. cruzi protein antigen fraction from epimastigotes bound to a nitrocellulose membrane was proposed as a supplemental test for Chagas' disease (See, Mendes, R. P., et al., J. Clin. Microbiol., 35:1829-1834 (1997)). A similar role has been proposed for an immunoblot assay based on a trypomastigote excreted-secreted antigen fraction (“TESA”) produced in cultures of T. cruzi-infected mammalian cells (See, Berrizbeitia, M., et al., J. Clin. Microbiol., 44:291-296 (2006), Umezawa, E. S., J. Clin. Microbiol., 34:2143-2147 (1996)). The biohazard inherent in manipulating cultures of live parasites and the difficulty of producing these complex antigen mixtures with lot-to-lot consistency are major disadvantages of assays based on antigens from culture such as these two. Additionally, none of these tests have been adopted as a confirmatory test and none has been developed commercially. Another assay, namely, the INNO-LIA Chagas assay, contains seven T. cruzi single-domain test bands on a plastic strip. However, the fact that seven distinct recombinant antigens are present on the strip could be a limiting factor in terms of its sensitivity. Moreover, having seven test bands complicates the interpretation protocol.
Thereupon, there remains a need in the art for an assay for T. cruzi that shows a high level of sensitivity and specification, that is simple to interpret and that is suitable for use as a confirmatory test for clinical and blood bank specimens that are borderline or reactive in screening assays.